Large scale genotyping of diseases and a diagnostic test for spinocerebellar ataxia type 6

ABSTRACT

The present invention provides a method of screening individuals at risk for developing diseases caused by trinucleotide repeat sequence instability. Specifically, the present invention is drawn to screening individuals at risk for developing autosomal dominant spinocerebellar ataxia type 6 by determining the length of a CAG trinucleotide repeat in the α 1A  calcium channel gene of the individual. In addition, there is provided a method of identifying genes which are disease-causing due to trinucleotide repeat sequence instability by large scale genotyping.

CROSS-REFERENCE TO RELATED APPLICATION

This is a continuation of application U.S. Ser. No. 09/093,273 filed onJun. 8, 1998, now abandoned.

This application is a divisional of U.S. Ser. No. 08/779,801, filed Jan.7, 1997 now U.S. Pat. No. 5,853,995.

FEDERAL FUNDING LEGEND

This invention was produced in part using funds obtained through a grantfrom the Department of the Army. Consequently, the federal governmenthas certain rights in this invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to the fields of moleculargenetics and diagnosis of genetic diseases. More specifically, thepresent invention relates to a large scale genotyping of diseases anddiagnostic tests and kits for same.

2. Description of the Related Art

Expansion of repeat sequences involving the trinucleotides CAG, CTG, CGGor GAA has been shown to be the primary cause of several neurologicaldisorders¹. Among them, CAG repeat expansions have been associated witha group of neurodegenerative disorders including Huntington disease²,spinobulbar muscular atrophy³, spinocerebellar ataxia type 1 (SCA1)⁴,spinocerebellar ataxia type 2 (SCA2)⁵⁻⁷, spinocerebellar ataxia type3/Machado-Joseph disease (SCA3/MJD)⁸, and dentatorubral-pallidoluysianatrophy/Haw-River syndrome⁹. All these disorders are progressivediseases leading to degeneration of the neurons in central nervoussystem. The CAG repeats in the respective genes show length polymorphismin the human population, typically, not exceeding 40 repeats. Inaffected individuals, the expanded alleles contain 36-121 repeats¹⁰.

CAG repeat expansions are much smaller than the hundreds or thousands ofrepeats often seen in diseases with CGG, CTG, and GAA expansions¹¹⁻¹⁴.The expanded CAG alleles show variable degrees of instability in bothgermline and somatic tissues^(15,16). Intergenerational changes of theCAG repeat size are often biased toward further expansion, particularlyif paternally transmitted, providing the molecular basis foranticipation. The CAG repeat arrays in these diseases are located in thecoding regions of the involved genes and are translated intopolyglutamine tracts in the protein products¹⁷. It has been postulatedthat an expansion of the polyglutamine tract produces a gain of functionin the protein product in each disease accounting for the dominantinheritance. Based on the relatively uniform characteristics of diseasescaused by CAG repeat expansions, it has been speculated that otherneurodegenerative diseases with similar clinical characteristics mayhave expansions of CAG repeats. Indeed, a study by Trottier andcolleagues demonstrated that an antibody against a polyglutamine tractdetects abnormally large proteins in tissues from patients with eitherSCA2 or spinocerebellar ataxia type 7 (SCA7), suggesting that themutation responsible for SCA2 and SCA7 is an expansion of apolyglutamine repeat tract¹⁸.

The prior art is deficient in the lack of effective means for the largescale genotyping of genetic diseases and diagnostic tests and kits fordiagnosing such diseases. The present invention fulfills thislong-standing need and desire in the art.

SUMMARY OF THE INVENTION

A polymorphic CAG repeat was identified in the human α_(1A)voltage-dependent calcium channel subunit. To demonstrate that expansionof this CAG repeat could be the cause of an inherited progressiveataxia, a large number of unrelated controls and ataxia patients weregenotyped. Eight unrelated patients with late onset ataxia had alleleswith larger repeat numbers (21-27) compared to the number of repeats(4-16) in 475 non-ataxia individuals. Analysis of the repeat length infamilies of the affected individuals revealed that the expansionsegregated with the phenotype in every patient. Six isoforms of thehuman α_(1A) calcium channel subunit were identified. The CAG repeat iswithin the open reading frame and is predicted to encode glutamine inthree of the isoforms. Thus, a small polyglutamine expansion in thehuman α_(1A) calcium channel is most likely the cause of a newlyclassified autosomal dominant spinocerebellar ataxia, SCA6.

In one object of the present invention, there is provided a method ofscreening individuals at risk for developing diseases caused bytrinucleotide repeat sequence instability, comprising the steps of:amplifying genomic DNA trinucleotide repeat sequences in a sample froman individual by polymerase chain reaction using one or moreoligonucleotide primers; restricting said amplified genomic DNAtrinucleotide repeat sequences with a restriction enzyme; separatingsaid restricted amplified genomic DNA trinucleotide repeat sequences byelectrophoresis to form a sample electrophoresis pattern; labeling aprobe capable of detecting said amplified genomic DNA trinucleotiderepeat sequences in said sample; hybridizing said sample of restricted,amplified genomic DNA trinucleotide repeat sequences with a firstaliquot of said labeled probe under hybridizing conditions to produce asample hybridization pattern for said sample genomic DNA trinucleotiderepeat sequence; amplifying a control genomic DNA trinucleotide repeatsequence by polymerase chain reaction using said one or moreoligonucleotide primers, wherein said control genomic DNA trinucleotiderepeat sequence is from non-diseased source; restricting said controlgenomic DNA trinucleotide repeat sequence with a restriction enzyme;separating said restricted control genomic DNA trinucleotide repeatsequence by electrophoresis to form a control electrophoresis pattern;combining said restricted control genomic DNA trinucleotide repeatsequence with a second aliquot of said probe under hybridizingconditions to form a control hybridization pattern for said genomic DNAtrinucleotide repeat sequence; comparing said sample hybridizationpattern for said sample genomic DNA trinucleotide repeat sequence tosaid control hybridization pattern for said control genomic DNAtrinucleotide repeat sequence; and determining whether said individualto be tested may be at risk for developing diseases caused bytrinucleotide repeat sequence instability, wherein if said samplegenomic DNA trinucleotide repeat sequence is larger than said controlgenomic DNA trinucleotide repeat sequence, said individual may be atrisk for developing diseases caused by trinucleotide repeat sequenceinstability.

In another object of the present invention, there is provided a methodof identifying genes in which a disease-causing allele is due totrinucleotide repeat sequence instability, comprising the steps of:screening a library with an oligonucleotide having a triplet baserepeat; identifying clones which have said triplet base repeat;sequencing said identified clones to determine sequences of nucleotidesflanking said triplet base repeat; synthesizing primers complementary tosaid sequences of nucleotides flanking said triplet base repeat;isolating DNA from a large sampling of individuals, including diseasedand non-diseased individuals; amplifying said isolated DNA with saidprimers to produce amplified triplet base repeat regions; determining anumber of triplet base repeats in said triplet base repeat region foreach of said individuals in said large sampling; determining whethertriplet base repeat expansions are observed at a relatively highfrequency in diseased individuals but are absent or occur at very lowfrequency in non-disease individuals, wherein if triplet base repeatexpansions are observed at a relatively high frequency in diseasedindividuals but are absent or occur at very low frequency in non-diseaseindividuals, it is likely that a disease-causing allele is due totrinucleotide repeat sequence instability.

Other and further aspects, features, and advantages of the presentinvention will be apparent from the following description of thepresently preferred embodiments of the invention given for the purposeof disclosure.

BRIEF DESCRIPTION OF THE DRAWINGS

So that the matter in which the above-recited features, advantages andobjects of the invention are attained and can be understood in detail,more particular descriptions of the invention may be had by reference tocertain embodiments which are illustrated in the appended drawings.These drawings form a part of the specification. It is to be noted,however, that the appended drawings illustrate preferred embodiments ofthe invention and therefore are not to be considered limiting in theirscope.

FIGS. 1A-1B show isoforms of the human α_(1A) voltage-dependent Ca²⁺channel. FIG. 1A shows that all the different isoforms have beenobserved in at least two independent cDNA clones. The “ ” represents a94 base pair nucleotide variation and the “ ” represents a 36 bpdeletion. The site of the GGCAG insertion is indicated by a vertical barand the position of the glutamine tract (poly Q) is shown as “ ”. Theamino acid changes affected by these variations are shown in FIG. 2.Only the isoforms with the GGCAG insertion have the extended openreading frame. FIG. 1B shows the sequences flanking the stop codon ofthe human Ca²⁺ channel isoforms BI-1 and BI-1 (GGCAG). The top andbottom letters indicate the respective amino acid encoded by thesequence. The stop codon is indicated by the TAN nucleotide. Thenucleotide “N” is a “G” nucleotide which has a decreased size of the “G”peak following an “A” peak, a characteristic of the FS Taq enzyme in dyeterminator sequencing chemistry from Applied Biosystem. It was confirmedthat this indeed is a “G” nucleotide when the reverse strand wassequenced. The complementary sequence of TAG, CTA is underlined.

FIGS. 2A-2C show the sequence comparison between the rabbit (BI-1) andhuman a₁ voltage-dependent Ca²⁺ channel. FIG. 2A shows the amino acidsequence from 1301 to 1840, FIG. 2B continues the amino acid sequencefrom 1841 to 2320, and FIG. 2C further continues the amino acid sequencefrom 2321 to 2528. The partial human cDNA sequence is a combination oftwo overlapping clones of 3.6 kb representing the largest deduced openreading frame. Identical amino acids are indicated by a “-” symbol andgaps in the alignment are represented by the “.” symbol. The human andrabbit BI-1 cDNAs share 90-94% amino acid identity depending on theisoforms. Since the full-length human α_(1A) voltage-dependent Ca²⁺channel has not determined, the amino acid strands in the rabbit BI-1sequence were numbered as reference (OCCCBI-1 in GenBank). Hypotheticalinsertion of the GGCAG nucleotides into the rabbit BI-1 isoform(accession No X57476) extends its deduced peptide reading frame by 237amino acids with the stop codon in the rabbit and human at identicalpositions. In this deduced reading frame the glutamine repeat isunderlined starting at amino acid position 2328 in the human and therabbit cDNA sequences. Without this insertion, the rabbit and human BI-1isoforms deduced reading frame stops at amino acid position 2273 asindicated by “*” (listed here as 2275 due to introduction of 2 alignmentgaps). The amino acids which vary in the isoforms corresponding to theV1, V2, V3 variations and GGCAG insertion are boxed. The V3 isoform hasa truncated 3′ region with a poly A+ tract. The sequences of therespective isoforms have been deposited in GenBank (accession numbers:U79663, U79664, U79665, U79666, U79667 and U79668).

FIG. 3 shows the northern analysis of human α_(1A) voltage-dependentCa²⁺ channel expression. Hybridization was carried out with the S-5 cDNAas probe. A distinct band of 8.5 kb was present in brain mRNA with asmear pattern specific to this probe and not detected using the β-actinprobe. The smearing in the mRNA from brain may reflect crosshybridization with the various alternative spliced forms or somedegradation.

FIGS. 4A-4D show the analysis of the PCR-amplified products generatedwith S-5-F1 and S-5-R1 primers flanking the CAG repeat in families withcerebellar ataxia. FIG. 4A shows the expanded allele with 27 repeats inthe four affected individuals (I.2, II.3, II.5, and II.7) from the INSCAkindred but in none of the asymptomatic family members. FIG. 4B showsthat the expanded allele of 22 CAGs repeats is observed in all fiveaffected members (II.1, II.2, II.3, III.1 and III.2) of the MS2SCAkindred. FIG. 4C shows that in the MDSCA kindred an aberrant size alleleof 23 CAG repeat was present in two brothers (II.1 and II.3) and asister (II.2) with clinical ataxia but not in the asymptomatic daughterof II.1. FIG. 4D shows the SISCA family where two affected members (IV.1and III.7) separated by five meiotic events share the same number of 22CAG repeats on their larger alleles. Tracing this allele through thepedigree indicates that their affected progenitors (III.5, II.2, II.4and I.2) most likely have this expanded allele.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method of screening individualsat risk for developing diseases caused by trinucleotide repeat sequenceinstability, comprising the steps of: amplifying genomic DNAtrinucleotide repeat sequences in a sample from an individual to betested by polymerase chain reaction using one or more oligonucleotideprimers; labeling a probe capable of detecting said amplified genomicDNA trinucleotide repeat sequences in said sample; combining said sampleof amplified genomic DNA trinucleotide repeat sequences with a firstaliquot of said labeled probe under hybridizing conditions to produce asample hybridization pattern for said sample genomic DNA trinucleotiderepeat sequence; amplifying a control genomic DNA trinucleotide repeatsequence by polymerase chain reaction using said one or moreoligonucleotide primers, wherein said control genomic DNA trinucleotiderepeat sequence is from non-diseased source; combining said controlgenomic DNA trinucleotide repeat sequence with a second aliquot of saidprobe under hybridizing conditions to form a control hybridizationpattern for said genomic DNA trinucleotide repeat sequence; comparingsaid sample hybridization pattern for said sample genomic DNAtrinucleotide repeat sequence to said control hybridization pattern forsaid control genomic DNA trinucleotide repeat sequence; and determiningwhether said individual to be tested may be at risk for developingdiseases caused by trinucleotide repeat sequence instability, wherein ifsaid sample genomic DNA trinucleotide repeat sequence is larger thansaid control genomic DNA trinucleotide repeat sequence, said individualmay be at risk for developing diseases caused by trinucleotide repeatsequence instability.

The present invention is additionally directed to a method ofidentifying genes in which a disease-causing allele is due totrinucleotide repeat sequence instability, comprising the steps of:screening a library with an oligonucleotide having a triplet baserepeat; identifying clones which have said triplet base repeat;sequencing said identified clones to determine sequences of nucleotidesflanking said triplet base repeat; synthesizing primers complementary tosaid sequences of nucleotides flanking said triplet base repeat;isolating DNA from a large sampling of individuals, including diseasedand non-diseased individuals; amplifying said isolated DNA with saidprimers to produce amplified triplet base repeat regions; determining anumber of triplet base repeats in said triplet base repeat region foreach of said individuals in said large sampling; determining whethertriplet base repeat expansions are observed at a relatively highfrequency in diseased individuals but are absent or occur at very lowfrequency in non-disease individuals, wherein if triplet base repeatexpansions are observed at a relatively high frequency in diseasedindividuals but are absent or occur at very low frequency in non-diseaseindividuals, it is likely that a disease-causing allele is due totrinucleotide repeat sequence instability.

In accordance with the present invention there may be employedconventional molecular biology, microbiology, and recombinant DNAtechniques within the skill of the art. Such techniques are explainedfully in the literature. See, e.g., Maniatis, Fritsch & Sambrook,“Molecular Cloning: A Laboratory Manual (1982); “DNA Cloning: APractical Approach,” Volumes I and II (D. N. Glover ed. 1985);“Oligonucleotide Synthesis” (M. J. Gait ed. 1984); “Nucleic AcidHybridization” [B. D. Hames & S. J. Higgins eds. (1985)]; “Transcriptionand Translation” [B. D. Hames & S. J. Higgins eds. (1984)]; “Animal CellCulture” [R. I. Freshney, ed. (1986)]; “Immobilized Cells And Enzymes”[IRL Press, (1986)]; B. Perbal, “A Practical Guide To Molecular Cloning”(1984).

Therefore, if appearing herein, the following terms shall have thedefinitions set out below.

A “vector” is a replicon, such as plasmid, phage or cosmid, to whichanother DNA segment may be attached so as to bring about the replicationof the attached segment. A vector is said to be “pharmacologicallyacceptable” if its administration can be tolerated by a recipientmammal. Such as agent is said to be administered in a “therapeuticallyeffective amount” if the amount administered is physiologicallysignificant. An agent is physiologically significant if its presenceresults in a change in the physiology of a recipient mammal. Forexample, in the treatment of retroviral infection, a compound whichdecreases the extent of infection or of physiologic damage due toinfection, would be considered therapeutically effective.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in either single stranded form,or a double-stranded helix. This term refers only to the primary andsecondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure herein according to the normal convention of giving only thesequence in the 5′ to 3′ direction along the nontranscribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

A DNA “coding sequence” is a double-stranded DNA sequence which istranscribed and translated into a polypeptide in vivo when placed underthe control of appropriate regulatory sequences. The boundaries of thecoding sequence are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and even synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence.

The term “oligonucleotide”, as used herein in referring to the probe ofthe present invention, is defined as a molecule comprised of two or moreribonucleotides, preferably more than three. Its exact size will dependupon many factors which, in turn, depend upon the ultimate function anduse of the oligonucleotide.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, the source of primerand the method used. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The labels most commonly employed for these studies are radioactiveelements, enzymes, chemicals which fluoresce when exposed to ultravioletlight, and others. A number of fluorescent materials are known and canbe utilized as labels. These include, for example, fluorescein,rhodamine, auramine, Texas Red, AMCA blue and Lucifer Yellow. Aparticular detecting material is anti-rabbit antibody prepared in goatsand conjugated with fluorescein through an isothiocyanate.

The following examples are given for the purpose of illustrating variousembodiments of the invention and are not meant to limit the presentinvention in any fashion.

EXAMPLE 1

Isolation of S-5 cDNA

The isolation of the S-5 cDNA was carried out by screening a primaryhuman brain CDNA library with a radiolabeled oligonucleotide probe(GCT)₇. The human brain cDNA was oligo-d(T) primed using Guber andHoffman methodology⁴⁴ with mRNA purchased from Clontech (Palo Alto,Calif.). The cDNA library was constructed with Not I restriction linkerfor cloning into IZAP II vector. The library was plated at a density of1000 plaques per 150 mm Luria broth agar plates. A total of 150,000primary clones were screened. Hybridization with a radiolabeledoligonucleotide probe (GCT)₇ was carried out at 55° C. using standardaqueous hybridization solution⁴⁵. The filters were washed 3 times for 30minutes each at 55° C. in 2×SSC and 0.1% SDS. Hybridizing clones werepurified for plasmid rescue. Plasmid DNAs was isolated using an AutoGen740 instrument and were sequenced using ABI kit and protocol on aABI-373A sequencer. Sequencing of the cDNAs were carried out to confirmthe presence of the triplet repeat sequence. The S-5 cDNA was one out ofthe 387 unique recombinant cDNAs obtained by this approach. Additionalclones of the α_(1A) calcium channel were isolated by using the S-5 cDNAas probe. In addition to the above human brain cDNA library, acommercial human fetal brain cDNA library with Eco RI cloning site fromStrategene (La Jolla, Calif.) was screened and the identified clonesfrom the library were used to reconstruct the 3′ region from the Not Isite to the poly (A) tract.

EXAMPLE 2

PCR Analysis

The degree of CAG length polymorphism in the α_(1A) calcium channel wasdetermined by the following primers: S-5-F1(5′-CACGTGTCCTATTCCCCTGTGATCC-3′) (SEQ ID NO:1) and S-5-R1(5′-TGGGTACCTCCGAGGGCCGCTGGTG-3′) (SEQ ID NO:2), though any appropriateprimers based on the sequence of the α_(1A) calcium channel gene may beused for this purpose. For each reaction, 5 pmol of each primer wasend-labeled with 1 mCi of [γ-³²P]ATP using 0.05 unit of polynucleotidekinase for 30 minutes. Each PCR analysis contained 20 ng of genomic DNAmixed with 5 pmol each of radiolabeled S-5-R1 and S-5-F1 primers in atotal volume of 25 ml containing 0.25 unit of Taq polymerase, 125 μMdNTP, 10 mM Tris pH 8.9, 2.5 mM MgCl₂, 30 mM KCl, and 3.5% (V/V)glycerol. The samples were denatured at 95° C. for 3 minutes, followedby 28 cycles of denaturation (94° C., 25 seconds), annealing (68° C., 30seconds) and extension (72° C., 2 minutes). Fifteen ml of formamideloading dye was added to the reaction, the mixture was denatured for 20minutes at 95° C. Seven ml were electrophoresed through a 6%polyacrylamide/8 M urea gel. Alleles sizes were determined by comparingmigration relative to an M13 sequencing ladder. Control DNAs usedincluded 65 samples from the CEPH families; 125 unrelated controlsprovided by various colleagues in the department of Molecular and HumanGenetics; 160 samples from diabetic sibling pairs; 41 sporadic breastcancer cases; 42 from Parkinson index cases; 24 from dystonia indexcases and 18 sporadic Alzheimer cases.

EXAMPLE 3

Northern Analysis

The northern blot containing poly A⁺ RNA from multiple human tissues waspurchased from Clonetech. 200 ng of S-5 cDNA insert was radiolabeledwith [α-³²P]dCTP using a random labelling kit from Pharmacia. The probewas hybridized overnight at 65° C. according to the protocol recommendedby Clonetech. The filter was washed 3 times at 68° C. for 30 minuteseach in 0.1×SSC, 0.1% SDS and then exposed to X-ray film. Lowerstringency washes at 68° C. at 0.5×SSC and 0.1% SDS gave many more bandsin different tissues suggesting cross reaction with other calciumchannel genes.

EXAMPLE 4

Linkage Analysis

Inspection of the genotype data shows a clear association between anincreased number of CAG repeats and the ataxia phenotype. Of the 133ataxia patients, eight had repeat lengths greater than 20, whereas noneof the controls had repeat lengths greater than 16. This association wasassessed statistically using a 2×2 table comparing the presence ofexpansions in ataxia cases versus controls. The level of significancewas determined using Fisher's exact test.

Haplotype analysis was used to show that the expansion and disease aretransmitted together. To model the situation of a single locus with botha phenotype (ataxia) and a polymorphism (expansion): two loci were used,one disease locus and one polymorphism, completely linked and incomplete linkage disequilibrium. The haplotype frequencies werecalculated by assuming all 133 of the cases suffer from some kind ofdominantly inherited ataxia. There should, therefore, be one diseasecausing mutation for each case. Eight of these mutations (approximately6%) were caused by CAG repeat expansions; the other 94% were caused byother mutations, either non-expansion mutations in this gene ormutations in other genes. The additional information needed to calculatethe haplotype frequencies is the population frequency of dominant ataxiaat unknown loci. The higher the estimate of this frequency the lower thelod scores. A conservative number of 1 in 500 was used for this analysiswhich places the gene frequency at 1 in 1000. The four haplotypefrequencies are then: 0.999 (no ataxia-no expansion), 0.0 (noataxia-expansion), 0.00094 (ataxia-no expansion), and 0.00006(ataxia-expansion). These haplotype frequencies were used to calculatethe lod scores in the four ataxia families using the FASTLINK version3.0P software programs. The affection status and genotypes were set forall patients, while unaffected and ungenotyped individuals werespecified as unknown affected status and unknown genotype.

To identify diseases which are caused by an expansion of a CAG repeat, alarge scale genotyping survey was performed on using polymorphic CAGrepeats and DNA samples from patients with late onset neurodegenerativediseases. The present invention reports that the human homolog of therabbit α_(1A) voltage-dependent calcium channel BI-1 gene contains apolymorphic CAG repeat sequence which is expanded in a fraction ofpatients diagnosed with autosomal dominant cerebellar ataxia. Theseresults indicate that the expansion of a CAG repeat predicted to encodefor polyglutamine in the human α_(1A) voltage-dependent Ca²⁺ channelgene is the apparent cause for one form of cerebellar ataxia.

EXAMPLE 5

CAG Repeats in the Human α_(1A)-Calcium Channel Subunit

To identify genes containing trinucleotide repeat sequences, anunamplified human brain cDNA library was screened using a (GCT)₇ repeatoligonucleotide as a probe. This screen identified 387 cDNA clonesdetermined to be independent based on sequence analysis. The repeatsizes in these clones ranged from 4 to 21. Partial cDNA clonescorresponding to the dentatorubral-pallidoluysian atrophy/Haw-River⁹ andMachado-Joseph disease⁸ genes were isolated in this screen. cDNA clonescorresponding to the SCA1, SCA2, and Huntington disease genes were notisolated in this screen, most likely because the CAG repeat in each ofthese genes is located in the 5′ region of a large transcript and thecDNA library screened is biased for 3′ cDNA termini given that it wasgenerated using oligo-d(T) priming.

The first clone examined extensively was a cDNA designated S-5 thatcontained 13 CAG repeats. The deduced peptide sequence of this 1.2 kbcDNA has 90% amino acid identity to the BI-1 isoform of the rabbitα_(1A) voltage-dependent Ca²⁺ channel (also known as P/Q-type Ca²⁺channel) suggesting that the S-5 clone is a partial cDNA of the humanhomolog¹⁹. The deduced human peptide sequence is also 90% identical tothe rat brain α_(1A) Ca²⁺ channel subunit²⁰. Partial human cDNA sequencecorresponding to rabbit BI-1 amino acid position 722-1036 was previouslyreported to share 92% and 82% with the rabbit and rat α_(1A) subunit ofcalcium channel, respectively²¹. The cDNA of the present inventioncontains coding sequence which corresponds to the carboxy terminusregion of the rabbit protein beginning at amino acid position 1325. Thesequence data suggest that the cDNA isolated encodes the human α_(1A)subunit of the calcium channel.

Using the somatic cell hybrid mapping panel #2 from Corriel, the α_(1A)Ca²⁺ channel was localized to human chromosome 19 by sequence tag site(STS) mapping. Diriong et al.²², have reported the mapping of the α_(1A)Ca²⁺ channel subunit to human chromosome 19p13 using a partial cDNAclone. The gene symbol of this locus was designated CACNL1A4²². Apartial human cDNA, (corresponding to rabbit BI-1 nucleotide position6487-7165) of the CACNL1A4 gene was reported by Margolis et al.²³ andwas shown to map to chromosome 19. A report describing the full-lengthsequence of the human CACNL1A4 gene was published recently by Ophoff andcollegues²⁴.

In rabbit, two isoforms (BI-1 and BI-2) of the α_(1A) calcium channelsubunit have been identified¹⁹. These isoforms differ from each other inthe carboxy terminus sequence where BI-2 has an additional 151 aminoacids. These isoforms are believed to result from an insertion-deletionof 423 nucleotides. The presence of the 423 nucleotides in BI-1introduces a stop codon which leads to the shorter, 2273 amino acidisoform. In rat brain, at least four alternatively spliced isoforms ofthe α_(1A) Ca²⁺ channel gene have been observed, but the sequence ofonly one isoform has been reported²⁰.

Comparison between the rabbit and human sequences revealed that the CAGrepeat was conserved and was located in the deduced 3′ untranslatedregion of the rabbit α_(1A) Ca²⁺ channel BI-1 and the S-5 cDNAs. Thefinding of a high degree of identity (84% identity over 700 nucleotides)between the 3′ untranslated region of the rabbit BI-1 isoform and thehuman S-5 clone of the present invention, raised the possibility thatadditional splice variants may occur and that some may contain an openreading frame in which the CAG repeat is translated. To examine this,the primary human cDNA library and a commercial fetal brain cDNA librarywas rescreened using the S-5 cDNA as probe. In total, 17 additionalclones were isolated, and careful sequence analysis of these clonesallowed identification of several alternatively spliced isoforms of thecarboxyl region of the human α_(1A) Ca²⁺ channel (FIG. 1A). Inparticular, five of these cDNAs contain a 5 base pair (GGCAG) insertionprior to the TAG stop codon of the S-5 cDNA (FIG. 1B). Clones with this5 base pair insertion have an extended deduced open reading frame of anadditional 239 amino acids in the human gene. Hypothetical insertion ofthis 5 base pair sequence into the rabbit BI-1 calcium channel at aminoacid position 2273 extends its deduced reading frame by 237 amino acids,and the peptide homology to the human sequence remains highly conserved(80% identity) arguing for the presence of such an isoform in the rabbitbrain (see FIG. 2). In this BI-1 (GGCAG) isoform, the CAG repeat encodesfor polyglutamine starting at amino acid position 2328 in the human andrabbit α_(1A) calcium channel gene.

Additional isoforms of the human α_(1A) Ca²⁺ channel gene were alsoobserved in the other clones. To ensure that none of these resulted fromcloning artifacts, at least two independent clones for each isoform wereisolated and sequenced. In total, six variants were observed includingthe variant identical to the rabbit BI-1 isoform also designated BI-1for human. The variant designated BI-1(V1) has a 94 base pair sequencewhich differs at the nucleotide level from BI-1 but is homologous at theamino acid level. This variant has also been described in rabbit¹⁹. TheBI-1(V1) isoform isolated in this study is 99.8% identical to thededuced peptide sequence described by Ophoff et al.²⁴. There are threedifferences involving amino acids at positions 1460 (Ala to Gly), 1605(Ala to Val), and 1618 (Ala to Val). The amino acids at these positionsin the deduced sequence are consistent in several clones analyzed andare identical to the rabbit and rat α_(1A) Ca²⁺ channel subunit deducedamino acids. The BI-1 and the BI-1(V1) isoforms are observed incombination with the GGCAG insertion (SEQ ID No. 3 and SEQ ID No. 4).Additional splice variants include BI-1(V2)-GGCAG (SEQ ID No. 5) whichhas a 36 nucleotide deletion and a variant with a truncated 3′ regionBI-1-(V2,V3) (FIG. 1A). The identified clones have differentcombinations of these variants with identical flanking sequences in thenon variant segment thereby ruling out cloning artifacts.

Consistent with the presence of multiple isoforms, northern analysis athigh hybridization stringency with the S-5 cDNA gave a single band of8.5 kb overlaying a smear above and below the predominant size mRNA inbrain (FIG. 3). At lower hybridization stringency, many additional bandswere observed in all tissues suggesting cross hybridization to othertypes of calcium channels (data not shown). All of the clones from thishuman brain library, which range from 1.2 to 3.1 kb in size, representonly the carboxyl region of the human α_(1A) Ca²⁺ channel subunit. TheCAG repeat in the respective adult brain cDNAs which were derived from asingle human mRNA source contained either 11 or 13 repeats, suggestingthe representation of polymorphic CAG alleles transcribed from thehomologous chromosome pair.

EXAMPLE 6

Large Scale Genotyping Survey for Expanded CAG Repeats

The possibility of identifying aberrant length CAG repeat sequencesdistinguishable from normal length polymorphism in the human α_(1A) Ca²⁺channel subunit was examined via a large scale genotyping survey ofataxia patients. This technique is based on the premise that iftrinucleotide expansion is responsible for SCA6, expansions would beobserved at a relatively high frequency in affected individuals butwould be absent or occur at very low frequency in non-disease alleles.

DNA samples from 475 unrelated non-ataxia individuals in the generalpopulation and 133 DNA samples from unrelated index cases known to haveprogressive cerebellar ataxia were analyzed. Using a pair ofradiolabeled synthetic oligonucleotide primers flanking the CAG repeatsequence of the human α_(1A) Ca²⁺ channel subunit, the CAG repeat regionof each sample was amplified and the size of the CAG repeat region wasdetermined via gel electrophoresis. The repeat sizes of the ataxia groupsamples were compared with those obtained from the DNA of the generalpopulation samples.

Table 1 shows the distribution of the CAG repeat sizes in the α_(1A)Ca²⁺ channel subunit gene of the 133 index patients with cerebellarataxia as well as the distribution of the CAG repeat sizes in the α_(1A)Ca²⁺ channel subunit gene of the 475 non-ataxia samples. The ethnicbackground of the control and patient populations included individualsof Caucasian, African American, Hispanic and Asian ancestry. Individualsfrom the general population displayed 10 alleles ranging from 4 to 16CAG repeat units and a heterozygosity of 71%. In the cerebellar ataxiapatients, the number of CAG repeats ranged in size from 7 to 27 with aheterozygosity of 74%. As can be seen in the allele size distribution,eight unrelated patients out of 133 ataxia index cases (6%) had a largersize allele of at least 21 CAG repeat units. Although the expansion wasrelatively small it was not observed in 475 individuals from thenon-ataxia controls making it extremely unlikely to be normal lengthpolymorphism (P<10⁻⁵ using Fisher's exact test).

TABLE 1 Comparison of the number of CAG repeat units on Ataxia andNon-Ataxia chromosomes Number of CAG Non-ataxia controls Ataxia indexcontrols repeat units Number of chromosomes Number of chromosomes  4 21 0  5 0 0  6 4 0  7 65  27   8 2 2  9 0 0 10 0 0 11 398  91  12 150  57 13 264  73  14 39  7 15 6 1 16 1 0 17 0 0 18 0 0 19 0 0 20 0 0 21 0 1 220 5 23 0 1 . . . . . . . . . 27 0 1

The genomic DNA from these eight index cases was amplified by the S-5primers, subcloned and sequenced. The number of CAG repeat unitsobtained from sequence analysis was consistent with an increase in thenumber of pure CAG repeat units in the α_(1A) Ca²⁺ channel subunit. Thedifferent number of CAG repeat units in these expanded alleles arguesagainst a rare founder allele. The observation of aberrant alleles ofexpanded sizes in the ataxia population and their absence in the generalpopulation was consistent with the possibility that these expandedalleles represent the mutational basis in a fraction of the ataxiapatients analyzed.

The method of large scale genotyping was effective in identifying theCAG expansin in the α_(1A) Ca²⁺ channel subunit gene. Thus, this conceptmay be used in the search for other mutation types associated withtriplet repeat disease phenomenon. Basically, one assumes thattrinucleotide repeat expansion is associated with alleles at highfrequency in disease phenotypes, but absent or at low frequency innon-disease phenotypes. Large scale genotyping, thus, is different fromthe approaches used for the identification of other human disease genes,including the positional cloning approach. In the positionaly cloningapproach, a genetic linkage to a specific chromosomal region must beestablished prior to the isolation of the candidate disease gene.Positional cloning was used for the identification of the genes forHuntington disease, spinobulbar muscular atrophy, spinocerebellar ataxiatype 1, spinocerebellar ataxia type 2, spinocerebellar ataxia type3/Machado-Joseph disease, and the genes associated with Fragile X andmyotonic muscular dystrophy.

The approach of the present invention also is different from randomcandidate gene approach for human disease, whereby no systematicstrategy is used in the identification of genes. The random candidategene approach was used in the identification of thedentatorubral-pallidoluysian atrophy/Haw-River syndrome gene. Thestrategy of the present invention is based on the observation thattriplet repeat sequences in disease genes are polymorphic in lengthwhich makes them suitable for a large scale genotyping survey. The largescale genotyping approach identifies aberrant allele sizes in diseasedindividuals as compared with the non-disease population. Thisconcept-driven strategy negates the need for prior establishment ofspecific genetic association (linkage) in family pedigrees as isemployed as a first step in a positional cloning. The large scalegenotyping strategy of the present invention is adirect-gene-to-disease-state approach.

In another object of the present invention, there is provided a methodof identifying genes in which a disease-causing allele is due totrinucleotide repeat sequence instability, comprising the steps of:screening a library with an oligonucleotide having a triplet baserepeat; identifying clones which have said triplet base repeat;sequencing said identified clones to determine sequences of nucleotidesflanking said triplet base repeat; synthesizing primers complementary tosaid sequences of nucleotides flanking said triplet base repeat;isolating DNA from a large sampling of individuals, including diseasedand non-diseased individuals; amplifying said isolated DNA with saidprimers to produce amplified triplet base repeat regions; determining anumber of triplet base repeats in said triplet base repeat region foreach of said individuals in said large sampling; determining whethertriplet base repeat expansions are observed at a relatively highfrequency in diseased individuals but are absent or occur at very lowfrequency in non-disease individuals, wherein if triplet base repeatexpansions are observed at a relatively high frequency in diseasedindividuals but are absent or occur at very low frequency in non-diseaseindividuals, it is likely that a disease-causing allele is due totrinucleotide repeat sequence instability.

EXAMPLE 7

Inheritance of Expanded Alleles in Ataxia Patients

Four of the index cases were from families where additional affectedmembers have been clinically evaluated, and DNA could be obtained forgenotypic analysis. Twenty-one family members participated in the studyafter informed consents were obtained. Fourteen of the 21 had clinicalevidence of ataxia. In each of these families, the ataxia was inheritedin an autosomal dominant manner with the age of onset ranging between 28and 50 years.

Genotypic analyses of family members using the S-5 primers demonstratedthat the expanded allele segregated with the disease phenotype in eachfamily. For example, FIG. 4A shows the expanded allele with 27 repeatsin the four affected individuals from the INSCA kindred but in none ofthe asymptomatic family members including a distantly related member(data not shown). In this kindred the age of onset ranged between 28 and31 years, and three of the asymptomatic individuals were 41 years old orolder. FIG. 4B shows that the expanded allele of 22 repeats was observedin all five affected members of the MS2SCA kindred. In the MDSCA kindred(FIG. 4C) an aberrant size allele of 23 CAG repeat was present in twobrothers (II.1 and II.3) and a sister (II.2) with clinical ataxia butnot in the asymptomatic daughter of II.1. In the SISCA family, shown inFIG. 4D, two affected members (IV.1 and III.7) separated by five meioticevents share the same number of 22 CAG repeats on their larger alleles.Tracing this allele through the pedigree indicates that their affectedprogenitors (III.5, II.2, II.4 and I.2) most likely have carried thisexpanded allele. The segregation of the expanded allele with the diseasein these families is highly significant as evident by a cumulativehaplotype lod score of 5.08 at a recombination frequency of zero whenthe genotypic data from affected individuals were analyzed using version3.0P of the FASTLINK computer programs (see above)^(26,27). The lodscores for each kindred are summarized in TABLE 2. Taken together, thestatistically significant finding that the expanded alleles are onlyobserved in patients diagnosed with cerebellar ataxia but not in 475non-ataxia controls and the clear cut association of these expandedalleles with disease demonstrate that the polyglutamine expansion in theα_(1A) voltage-dependent Ca²⁺ channel subunit is the cause of this lateonset dominantly inherited ataxia.

TABLE 2 Lod scores from haplotype analysis Family Lod score at Theta = 0INSCA 1.20 MDSCA 0.90 MS2SCA 1.49 SISCA 1.49 SUM 5.08

EXAMPLE 8

Clinical and Pathological Findings in Patients with CAG Repeat Expansion

The clinical features of the patients in the above-described familieswere very similar and consist predominantly of mild but slowlyprogressive cerebellar ataxia of the limbs and gait, dysarthria,nystagmus, and mild vibratory and proprioceptive sensory loss. Thedisease is very insidious and most patients do not realize they areaffected initially but do describe a sense of momentary imbalance and“wooziness” when they take a quick turn or make a rapid movement.Typically, it is years after this initial sensation when the patientsrealize that they have developed permanent balance and coordinationdifficulties. The disease usually progresses over 20-30 years leading toimpairment of gait and causing the patient to become wheel-chair bound.In the few older patients, choking has been observed suggestinginvolvement of the brain stem, and the disease has been the cause ofdeath in several members of the MDSCA and MS2SCA kindreds. Symptomsdevelop generally when the patients are in their forties in the MDSCA,SISCA, and MS2SCA families where the repeat number is 22-23; however inthe INSCA kindred where the expanded allele contains 27 repeats, thedisease onset is between 28 and 31 years in all the affectedindividuals. Magnetic resonance imaging of the brain in affectedindividuals reveals isolated cerebellar atrophy. Detailedneuropathologic studies on two deceased members from the SISCA kindredshowed marked cerebellar atrophy and very mild atrophy of the brainstem²⁸. Microscopic examination revealed severe loss of cerebellarPurkinje cells, moderate loss of granule cells and dentate nucleusneurons, and mild to moderate neuronal loss in the inferior olive.

The hereditary cerebellar ataxias are a clinically and geneticallyheterogenous group of neurological disorders associated with dysfunctionof the cerebellum and its afferent and efferent connections. To date,six autosomal dominant spinocerebellar ataxias (SCAs) have been mappedto human chromosomes 6, 12, 14, 16, 11, and 3 with the loci designatedSCA1, SCA2, SCA3, SCA4, SCA5, and SCA7, respectively¹⁰. The map locationof the genes in many families with dominantly inherited and progressiveataxias remains unknown. The mapping of the α_(1A) Ca²⁺ channel subunitto human chromosome 19p13 and the identification of the CAG repeatexpansion in this channel as the mutational mechanism in four familiesdefine a new SCA locus on human chromosome 19p13 which can be designatedSCA6.

In the past, the term SCA6 has been used to described dominantlyinherited SCAs that did not map to any of the known loci^(29,30). Thismapping nomenclature was revised to assign the SCA6 locus to thedominantly inherited ataxia mapping to chromosome 19p13 (HGMNomenclature Committee). Hereditary paroxysmal cerebellar ataxia (HPCA)or episodic ataxia (EA) has also been mapped to the 19p13 region³¹⁻³².The locus for another episodic disease, familial hemiplegic migraine(FHM)³³, has been localized to 19p13 in the region where the gene forHPCA/EA was assigned. Patients with HPCA or EA typically have periodicataxia with apparently normal coordination between attacks. This isreminiscent of the episodic sensation of unsteadiness described inpatients years before the ataxia becomes a permanent finding. The onlypersistent abnormality on neurologic exam in HPCA/EA is the presence ofnystagmus, a finding seen in all the patients. Brain imaging studiesrevealed that some HPCA/EA patients have cerebellar atrophy³¹.Interestingly, in several families with FHM, affected members have showndegenerative cerebellar atrophy which is associated with ataxia,nystagmus and other vestibulocerebellar ocular abnormalities, similar tothose seen in HPCA/EA³⁴. The overlap in the phenotypes of these twodisorders led to the hypothesis that HPCA/EA and FHM are allelicdisorders possibly caused by a mutation in an ion channel gene becauseof the periodic nature of the symptoms^(32,34).

Recently, Ophoff et al. reported four missense mutations in the humanα_(1A) Ca²⁺ channel subunit gene in families with FHM and two mutationsdisrupting the reading frame of the same gene in two families with EA²⁴.These results and the present invention demonstrate that FHM, HPCA/EAand the progressive SCA6 are allelic disorders. The nature of themutation (CAG repeat expansion in SCA6 versus protein truncation inHPCA/EA) affects the clinical course of the disease. Permanent andprogressive cerebellar and brain stem dysfunction were observed in SCA6whereas mild and intermittent cerebellar dysfunction was seen inHPCA/EA. This suggests that the glutamine expansion affects the functionof the channel in a manner which triggers progressive neuronal loss.This may be via alteration of neurotransmitter release or by causingabnormal levels of intracellular Ca²⁺ leading to subsequent celldeath^(21,35). At this time the pathogenic effects of each of thesemutations with regard to periodic neurological dysfunction versuspermanent and progressive disease cannot be determined and will have toawait transgenic mouse models and neurophysiologic studies. Althoughother mutations in the CACNL1A4 gene in SCA6 families has not beenexcluded, the highly significant association between expansion anddisease phenotype (P<10⁻⁵) in eight independent ataxia families and thedifferent number of repeats on expanded alleles in four families (in theabsence of intergenerational instability) argue strongly that this isthe disease causing mutation. It is also important to note that Ophoffand collegues²⁴ did not observe any expanded alleles in the 50 normalindividuals they genotyped.

Although the mutational mechanism in SCA6 proved to involve an expansionof a translated CAG repeat like the other dominantly inheritedprogressive ataxias, it is not clear whether the pathogenic mechanism issimilar. There are two key differences between the mutation in SCA6 andthose causing SCA1, SCA2, SCA3, HD, DRPLA, and SBMA. First, the expandedmutant alleles in SCA6 (21-27 repeats) are remarkably smaller than theexpanded alleles seen in any of the other neurodegenerative diseases(36-121 repeats) and are well within the normal range of polyglutaminetracts seen at the other loci in many unaffected individuals. Second,the CAG repeat expansion occurs in the coding region of a gene which isknown to be important for normal Purkinje cell function andsurvival^(19,25). This raises the possibility that the CAG expansion isexerting its pathogenic effect by directly interfering with the normalfunction of the α_(1A) calcium channel.

Voltage-dependent calcium channels mediate the entry of calcium intoneurons and other excitable cells and play important roles in a varietyof neuronal functions, including membrane excitability, neurotransmitterrelease, and gene expression³⁶. Calcium channels are multisubunitcomplexes with the channel activity mainly mediated by the pore-forminga₁ subunit, however, additional subunits including b, a₂/d, and g act asaccessory proteins that regulate channel activity³⁶⁻³⁸. The cDNAsencoding six a₁ genes have been cloned and have been designatedα_(1A,B,C,D,E) and S³⁹. The human gene characterized in the presentinvention is most homologous to the rabbit and rat α_(1A)isoforms^(19,20). The mapping assignment to human chromosome 19 isconsistent with the previous mapping of the human sequence encoding theα_(1A) isoform to chromosome 19p13²²⁻²⁴. A combination ofelectrophysiologic and pharmacologic properties define four main typesof high-threshold calcium channels in peripheral and central neurons ofmammals⁴⁰. These are designated L, N, P, and Q, with the P-type channelsbeing the predominant calcium channel in Purkinje cells, and the Q typebeing a prominent calcium current in cerebellar granule cells^(25,38).The cloned α_(1A) isoform has been shown to give rise to P and/or Q typecalcium currents^(38,40). The additional isoforms identified may helpresolve some of the functional differences observed for the P/Q typecalcium currents. The pharmacologic as well as the electrophysiologicproperties of the α_(1A) channel, together with its abundant expressionin rat cerebellum emphasize its importance for calcium entry andhomeostasis in Purkinje cells^(25,41).

Recently, the mouse homolog of the a_(1A) voltage-dependent subunit genehas been identified using a positional cloning strategy aimed atidentifying the gene mutated in the tottering (tg) and leaner (tg^(la))mice which show seizures and cerebellar ataxia⁴². This locus maps tomouse chromosome 8 in a region syntenic with human 19p13. The tgmutation, a C to T change at position 1802, causes a nonconservedproline to leucine substitution in a position very close to theconserved pore-lining domain in the extracellular segment of the secondtransmembrane domain. This mutation leads to a recessive neurologicaldisorder with ataxia, motor- and absence-type seizures.

The tg^(la) mutation is a single G to A change in the splice donorconsensus sequence at the 5′ end of an intron located in the C-terminusintracellular domain. This mutation gives rise to two aberrantly splicedmRNAs detected by RT-PCR; a larger fragment resulting from failure tosplice out the intron and a smaller fragment resulting from skipping ofone exon. Both transcripts are predicted to shift the reading frame andproduce abnormal proteins. Homozygous tg^(la) mice, which have thesplice mutation have more profound ataxia and cerebellar degenerationcompared to the tg mice.

The findings that mutations in the α_(1A) Ca²⁺ channel are associatedwith cerebellar ataxia and Purkinje and granule cell degeneration in themouse support the hypothesis that this channel is critical for normalPurkinje and granule cell function in the cerebellum. The recessivenature of the two mutations in the mouse and the fact that the tg^(la)mutation is predicted to generate an abnormal protein suggest that thesemutations are causing the ataxia phenotype through a loss of functionmechanism. The mutation in the tg^(la) mice alter the carboxy terminusportion of the channel just up stream from the position of the putativeglutamine tract in the human gene. These data raise interestingquestions about the mechanism by which a modest glutamine expansion inthe human α_(1A) Ca²⁺ channel isoform leads to the cerebellardegeneration and ataxia. The dominant nature of the disease wouldsuggest three possibilities: (1) loss of function due tohaploinsufficiency, (2) a dominant negative effect due to the expansion,or (3) a novel gain of function as has been suggested in other diseasescaused by CAG repeat expansions. The lack of ataxia phenotype in the tgand tg^(la) mice heterozygous for the mutation would argue against theloss of function hypothesis. However, this model can not be ruled outuntil it is confirmed that either mutation in the mouse truly leads to aloss of the α_(1A) Ca²⁺ channel function and that the heterozygous micedo not display ataxia nor Purkinje cell degeneration using carefulquantitative measures. Given the transient and mild nature of the ataxiain some of the patients it could be extremely difficult to ascertain amild and intermittent ataxia phenotype in the mice. A model invoking adominant negative mechanism is compatible with the inheritance patternin the human families and with data available sofar on the tg mice. Inthis model, the small expansion of the glutamine tract could interferewith the normal function of the channel either by affecting its bindingto synaptic proteins or by hindering its association with otheraccessory channel proteins that are known to modulate its activity.Given that the α_(1A) Ca²⁺ channel is now known to be important fornormal Purkinje cell function based on electrophysiologic data⁴³ and thedata in the tg mice, it is hard to argue that the glutamine expansion isconferring novel gain of function on the protein. The glutamineexpansion most likely leads to aberrant channel function including thepossibility of constitutive activation. The ultimate proof of thevarious models will await the generation of mice which lack the α_(1A)Ca²⁺ channel gene and mice which express an allele with a CAG expansionin the SCA6 disease range.

The genotype/phenotype correlation in SCA6 suggests that the expansionis quite deleterious given the dramatic difference in the age of onset(28-31 years) in every member of the family carrying the 27 repeats ascompared to the other families (40-50 years) when the repeat size is inthe 22-23 repeat range. Although the sample size is too small at thistime to draw firm conclusion about genotype/phenotype correlation, itwould be interesting to see if some patients with HPCA/EA, which is muchmilder than SCA6, would have even smaller expansions. In addition, itwould be important to determine if different mutations in the α_(1A)Ca²⁺ channel lead to SCA6. The CAG repeat in SCA6 is stable withoutdetectable mosaicism or intergenerational allele size changes. This isnot surprising given that similar size CAG repeats at many other locihave been shown to be transmitted in a stable manner. However, the sizeof the repeat in the general population and the different sizes ofexpanded alleles in different SCA6 families suggest that some degree ofinstability does occur at this locus and that such instability hasresulted in mutational expansions into the disease allele range.

In conclusion, the present invention demonstrates that a relativelysmall polyglutamine expansion in the human α_(1A) subunit of a Purkinjecell type Ca²⁺ channel leads to Purkinje cell degeneration andcerebellar ataxia. The immediate implications of this finding are bothclinical and biological. The observation that a relatively small CAGrepeat expansion can lead to abnormal protein function provides a newconcept about the effects of such repeats and the need to evaluate eachcarefully for possible pathogenic effects. Lastly, the expansion of apolyglutamine tract in a human calcium channel should provide insightabout mechanisms of neurodegeneration as they pertain to calciumhomeostasis and the possible role of such mechanisms in otherglutamine-mediated neurodegenerative processes.

The following references were cited herein:

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Any patents or publications mentioned in this specification areindicative of the levels of those skilled in the art to which theinvention pertains. These patents and publications are hereinincorporated by reference to the same extent as if each individualpublication was specifically and individually indicated to beincorporated by reference.

One skilled in the art will readily appreciate that the presentinvention is well adapted to carry out the objects and obtain the endsand advantages mentioned, as well as those inherent therein. The presentexamples along with the methods, procedures, treatments, molecules, andspecific compounds described herein are presently representative ofpreferred embodiments, are exemplary, and are not intended aslimitations on the scope of the invention. Changes therein and otheruses will occur to those skilled in the art which are encompassed withinthe spirit of the invention as defined by the scope of the claims.

1. A method of determining whether an individual has or is at risk fordeveloping a spinocerebellar ataxia, wherein the method comprisesanalyzing a CAG repeat region of an alpha_(1A) calcium channel subunitgene of said individual to detect a length polymorphism therein, whereina length polymorphism of greater than 20 CAG repeat units indicates thatsaid individual has or is at risk for developing a spinocerebellarataxia.
 2. The method of claim 1, wherein said analyzing the CAG repeatregion comprises amplifying by polymerase chain reaction said CAG repeatregion present in genomic DNA obtained from said individual using atleast one oligonucleotide primer capable of amplifying said CAG repeatregion, to produce an amplified CAG repeat region.
 3. The method ofclaim 2, wherein said oligonucleotide primer is labeled.
 4. The methodof claim 3, wherein said oligonucleotide primer is fluorescentlylabeled.
 5. The method of claim 4, wherein said fluorescent label isfluorescein, rhodamine, auramine, Texas Red, AMCA blue, or Luciferyellow.
 6. The method of claim 3, wherein said oligonucleotide primer isradiolabeled.
 7. The method of claim 6, wherein said radiolabelcomprises ³²P.
 8. The method of claim 3, wherein said oligonucleotideprimer is labeled with an enzyme.
 9. The method of claim 3, wherein saidoligonucleotide primer is single stranded.
 10. The method of claim 3,wherein said oligonucleotide primer is double stranded.
 11. The methodof claim 3, wherein said oligonucleotide primer comprises from between15 to 25 nucleotides.
 12. The method of claim 2, wherein saidoligonucleotide primer comprises SEQ ID NO:1.
 13. The method of claim 2,wherein said oligonucleotide primer comprises SEQ ID NO:2.
 14. Themethod of claim 2, further comprising fractionating said amplified CAGrepeat region by gel electrophoresis.
 15. The method of claim 14,wherein the size of said amplified CAG repeat region is measured usingsaid gel electrophoresis.
 16. The method of claim 1, wherein saidanalyzing the CAG repeat region comprises measuring the size of the CAGrepeat region.
 17. The method of claim 1, wherein said analyzingcomprises comparing the number of CAG nucleotide repeat units present insaid CAG repeat region to a control sample.
 18. The method of claim 17,wherein said control sample comprises a sequencing ladder.
 19. Themethod of claim 17, wherein said control sample is from a subject whodoes not have SCA-6.
 20. The method of claim 17, wherein said controlsample comprises DNA or RNA from a subject who does not have SCA-6. 21.The method of claim 17, wherein said control sample was derived from asubject who does not have SCA-6.
 22. The method of claim 1, wherein themethod comprises obtaining a biological sample from said individual. 23.The method of claim 22, wherein the biological sample is a blood sample.24. A method of determining whether an individual has or is at risk fordeveloping a spinocerebellar ataxia, wherein the method comprisesanalyzing an allele of an alpha_(1A) calcium channel subunit gene ofsaid individual to detect a CAG repeat region length polymorphismtherein, wherein a length polymorphism of greater than 20 CAG repeatunits indicates that said individual has or is at risk for developing aspinocerebellar ataxia.
 25. A method of detecting the presence orabsence of a CAG trinuncleotide repeat expansion comprising analyzing aCAG repeat region of an alpha_(1A) calcium channel subunit gene in agenomic DNA sample to detect a length polymorphism therein, wherein anumber of CAG repeat units greater than 13 indicates the presence of atrinucleotide repeat expansion.